1. Field of the Invention
This invention relates to an analysis of biologic fluids and, more particularly, this invention relates to the spectrophotometric analysis of albumin in biologic fluids using dye binding methods wherein the color imparting dye exhibits species specificity.
2. Description of the Prior Art
Spectrophotometric methods are widely used for determining the concentration of various constituents in biologic fluids such as blood. The methods usually involve adding a color-forming reagent, generally in aqueous solution, to the fluid to form a colored complex with the analyte to be detected.
The complex has a characteristic wavelength of maximum light absorption. By exposing a sample of the fluid containing the complexed reagent to a light source of the wavelength of maximum absorption (or the wavelength of maximum difference of absorption with respect to the reagent in a blank), and thereafter measuring the degree of light absorption, the concentration of the analyte in the biologic fluid can be determined with reference to a calibration graph constructed from absorption measurements made on calibrator solutions comprising standard solutions containing the reagent and known analyte concentrations.
The absorbance of both the sample and the standard are generally read against a blank containing the reagent solution. Of course, to accomplish the determination, a color-forming reagent must be selected which has the ability to form a colored derivative with the analyte, which derivative differs from the reagent in either intensity or absorption wavelength characteristics.
With respect to the determination of albumin in sera, several general types of dyes are useful as components of color-forming reagents. These include the sulphonphthalein dyes such as bromcresol green, hydroxyphenyl azo dyes such as 2-(hydroxyphenylazo)-benzoic acid (commonly called HABA), and biuret type reagents.
A dye marketed by Pierce Chemical Company of Rockford, Ill. (now Lancer Division, Sherwood Medical Industries Inc.) under the mark "Spectru AB2" is quite useful. A particularly advantageous albumin determination procedure employing sulphonphthalein dyes is described in Gindler U.S. Pat. No. 3,884,637 issued May 20, 1975. HABA and SpecTru AB2.TM. dyes have been used for determination of albumin for a number of years. Procedures using biuret are most useful when total protein (albumin plus globulin) is to be determined, since biuret forms colored complexes with both of these proteins. When biuret is to be used for albumin determination alone, it is necessary to first remove globulin from the sample.
There are many instances when it is desirable to determine globulin in addition to albumin concentration. These determinations are frequently accomplished by determining albumin with an albumin-binding color reagent and then using biuret reagent to determine the total protein concentrations. By difference, the globulin concentration can then be ascertained.
Some albumin-binding dyes exhibit species specificity with respect to albumin. Thus, whereas bromcresol green and biuret reagents display little difference in their binding capacities with respect to either human or bovine albumin, SpecTru AB2.TM. dye exhibits a more pronounced capacity to bind human albumin than bovine albumin.
As a result, when albumin concentration is to be determined in human sera using such a dye, it has heretofore been necessary to also use human albumin in the standard. However, the availability of pure human albumin has become irregular, in part because sources have sometimes provided human albumin potentially carrying hepatitis. On the other hand, suitable bovine albumin is generally available in good supply.